Osteoporotic fractures occur in 13-22% of men and 40-50% of women. Testosterone (T) replacement may hold promise for combating osteoporosis in males. The bone-protective effects of T may be mediated by conversion of T to estradiol by bone aromatase. Recently, young men have been identified with constitutive aromatase deficiency. These patients suffer from an osteopenia that responds to estrogen, but not T. A popular hypothesis holds that a major portion of the effects of T on bone are mediated by aromatase. We have recently made the 1st measurements of sex steroid hormones in bone with unexpected results. 1. In bone of male and female rats, T and DHT concentrations are far in excess of estradiol. While bone estradiol is low, estrone and estrone sulfate are abundant. T and DHT concentrations are ~100- fold higher in bone than in blood, while estradiol is lower in bone. 2. T administration increases cancellous bone volume and bone mechanical strength in females and males. 3. In both sexes, T administration elevates bone T and DHT, but not estradiol. Our revised hypothesis is that many of the effects of T on bone are mediated by aromatase, but that the estradiol formed is rapidly converted to other forms of estrogen. Our overall purposes are to understand the roles of T and estradiol metabolism in mediating the effect of T on bone and to provide a rationale for testing strategies to combat osteoporosis. Our first aim is to characterize blood and bone concentrations of sex steroids in adult and older men and women. We will determine 1) whether the rank order of concentration observed in rats [T >DHT >>>estradiol] also occurs in humans, 2) whether bone contains significant depots of estrone, estriol or conjugated estrogens and 3) the effect of aging on bone sex steroids. Our second aim is to explore, in animal models, the roles of estrogens in mediating the effects of T on bone. To characterize the role of aromatase in mediating the effects of T on bone, we will take two approaches 1) administration of T 1 vorozole to rats and 2) administration of T to aromatase knockout (ArKO) mice. To characterize the role of the enzyme 17-beta HSD1 in mediating the effects of T on bone, we will administer to rats T 1 B10720511. Vorozole may block T-induced bone anabolic effects (inhibition of resorption, increased cancellous bone mass assessed by histopmorphometry and bone mechanical strength). B10720511 may block or enhance the effects of T. PUBLIC HEALTH RELEVANCE: The loss of sex steroid hormone plays an important role in the losses in muscle and bone that accompany aging in men and women. The proposed experiments seek to determine the role played by bone metabolism in mediating the effects of testosterone on bone. The findings will help to guide the rational design of treatments to prevent osteopenia in the elderly.